Mapping the micro-proteome of the nuclear lamina and lamin associated domains

Abstract

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, so-called lamin associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions.In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

Lamin C regulates genome organization after mitosis

“…This led us to question whether lamin C might co-localize with LADs at the end of mitosis and into early G1. To test this we used a LAD-tracer system to fluorescently identify all endogenous LADs in living cells (MEFs) co-expressing EYFP-lamin C 9,16,53 . The LAD-tracer system relies on the expression of two constructs.…”

“…marks LADs with me A), but has two additional domains that strictly control its expression: a destabilization domain (DD) that causes degradation in the absence of the shield ligand, and a Cdc10 dependent transcript 1(CDT) regulatory domain restricts its expression to G1 9,16,53,54 . The second construct, the LADtracer, is a modified mCherry-tagged version of the previously described m6A-tracer 16,53 that binds me A, the modification generated by Dam-Lamin B1, thus marking LADs with mCherry. The LAD-tracer is expressed throughout the cell cycle and identifies me A-modified DNA (LADs) in all phases in cells where both constructs are expressed at appropriate levels.…”

“…LADs from murine embryonic fibroblasts were defined through the LADetector algorithm, and complementary regions to Chromosomes 11 and 12 were defined as non-LADs 9,16,53,54 . Data provided Geo GSE56990.…”

“…Immortalized C57Bl/6 MEFs (ATCC CRL-2752) cells were infected to stably express ddDam-LaminB1-CDT, EYFP-Lamin C and m6A tracer. ddDam-LaminB1-CDT is a destabilized version of the previously described DamID construct that has incorporated the CDT domain from the Fucci system to ensure its expression is restricted to interphase 9,16,53,54 . The m6A-tracer is comprised of a catalytically inactive version of DpnI, that retains its ability to bind DNA, in frame with an mCherry red fluorescent protein 16,53 .…”

“…ddDam-LaminB1-CDT is a destabilized version of the previously described DamID construct that has incorporated the CDT domain from the Fucci system to ensure its expression is restricted to interphase 9,16,53,54 . The m6A-tracer is comprised of a catalytically inactive version of DpnI, that retains its ability to bind DNA, in frame with an mCherry red fluorescent protein 16,53 . For cell cycle experiments, these cells were grown in the presence of shield ligand (AOBIOUS, AOB6677), which stabilizes the ddDam-LaminB1-CDT, along with 1mM thymidine (Sigma) block for 24 hours to enable synchronization of cells at G1/S.…”